A variety of chemical reactions are improved through the addition of heat. Increased temperature is of particular importance in certain biochemical reactions such as nucleic acid hybridizations where heat is frequently used to increase the specificity of the binding reaction. Elevated temperatures (i.e., greater than 70.degree. C.), are also useful in overcoming problems of secondary RNA structures in the reverse transcriptase reaction (RTR), used to produce complementary DNA (cDNA) from RNA (Biochemistry 30, 7661-7666, 1991). Elevated temperatures (i.e., greater than 70.degree. C.), are also useful in DNA sequencing procedures.
Heat is being employed in nucleic acid amplification such as the polymerase chain reaction (PCR). The PCR (U.S. Pat. Nos. 4,683,202 and 4,683,195) employs a heating and cooling cycle to drive the reaction. First, the reaction mixture is heated to, or above, the nucleic acid melting temperature (denaturization), then cooled to allow specific oligonucleotide primers to bind to the sample (annealing), and then heated to optimize the addition of complementary bases to the amplified nucleic acid (extension). Using heat stable, Taq DNA polymerase (U.S. Pat. No. 4,889,818), this cycle of denaturing, annealing and extension is repeated as many times as needed to generate the desired product.
The PCR is quickly becoming a major tool in molecular biology, and the need for high specificity during amplification is an increasing problem.
One method suggested for increasing the PCR specificity is called "booster" PCR (see application section below), where initial amplification is done with diluted amounts of primer, that are subsequently increased or "boosted" for later cycles.
Another method for increasing specificity is pre-amplification heating. This method has been described by H. A. Erlich, et al., Science 252, 1643-1651 (1991), and R. T. D'Aquila, et al., Nucleic Acids Res. 19, 3749 (1991). It requires exclusion of at least one essential reagent (dNTP's, Mg.sup.2-, DNA polymerase or primers), from the reaction until it has been heated to the desired annealing temperature. However, the procedure requires the sample tubes to be closed while heating, and reopened for addition of the missing reagent.
The procedure, also called Hot-Start PCR by Perkin Elmer Cetus, Conn., has been improved by the use of a wax barrier formed in the reaction tube that separates some of the reagents until the tube is heated to melt the barrier.
Perkin Elmer Cetus now sells a wax pellet (Ampliwax.TM.) for this purpose. Their published procedure includes the following steps: (1) to a sample tube containing primers, Mg.sup.2-, dNTP's in buffer, add one pellet of Ampliwax.TM.; (2) heat the tube to 80.degree. C. to melt the wax; (3) cool to room temperature to form a wax barrier on top of the solution; (4) over the wax barrier, add the sample with DNA polymerase in buffer; and (5) start normal PCR cycling.
The problems with this method are: (1) it is limited to open tubes and is not readily used in other containers such as sealed capillaries or the tubular containers described in the inventor's Disclosure Document No. 291836 (this disclosure relates to tubing with constantly heated or cooled zones where PCR is done by moving a liquid sample between the zones to heat and cool it); (2) there is potential for error in forming each wax barrier in each tube; and (3) the melting and cooling of wax adds additional steps to the PCR method.
The uses for Ampliwax.TM. wax pellets and greases to form barriers between reagents in a reaction tube are more fully disclosed in international publication number WO 91/12342, international patent application number PCT/US91/01039, to Cetus Corporation (hereinafter referred to as the "Cetus application").
The Cetus application is directed to the use of wax or grease as a substitute for oil in forming a vapor barrier over aqueous samples in a PCR reaction tube. Wax or grease is cast as a molten layer over an aqueous solution and allowed to cool and solidify. This layer can also be used as a barrier between subsets of PCR reagents within the reaction tube.
On page 8 of the Cetus application, there is a suggestion for PCR reagents "wherein one subset is incorporated into a lighter-than-water oil, grease, or wax which is layered on top of any aqueous suspension or solution." This is referring to oil, grease or wax that is layered over an aqueous solution to form a barrier in the reaction tube. At page 26, the Cetus application teaches that a wax or grease pellet added to an aqueous solution in a PCR tube is heated to melt the wax and form a barrier over the aqueous solution.
The Cetus application suggests that oil solubility and water extraction are potential obstacles of incorporating PCR reagents in wax or grease. Apparently, these problems arise because a hydrophobic barrier layer is needed to separate aqueous reagents within a PCR reaction tube. Low oil solubility can be a problem for reagents suspended in grease or wax. The Cetus application suggests using a water-wax or water-grease emulsion to incorporate PCR reagents in wax or grease. Only one reagent, magnesium salts of fatty acids, is disclosed as being suitable for incorporation into wax or grease in the absence of emulsification. At page 25, the Cetus application teaches that the molten layer of grease or wax must be cast and solidified quickly before the reagent is extracted into the aqueous layer below. To minimize extraction during casting of the barrier layer, the Cetus application recommends using "wax or grease with the lowest practical melting point, preferably in the range of about 40.degree.-50.degree. C."
It will be appreciated that there is a need in the art for methods of performing the PCR as well as in other high temperature methods such as reverse transcriptase and nucleic acid hybridizations, which are not limited to open tubes and which do not require the additional steps of forming a wax barrier in each tube.